Biological/bioactive factors (BFs), including growth factors, differentiation factors, cytokines and chemokines are crucial in maintaining normal tissue homeostasis, and wound healing and tissue regeneration. This is also the case for the skeletal tissues including the cartilage covering the surface of the joint (articular cartilage), meniscal cartilage in the joint and tendons, ligaments and bone. Damage to skeletal tissues can be caused through trauma, for example, but not exclusively, torn ligaments and tendons and bone fractures. Damage to skeletal tissues can also be caused by degenerative diseases. One example is a degenerative arthritis such as osteoarthritis, which can particularly affect articular cartilage and subchondral bone. Osteoarthritis is the most common form of arthritis' causing painful joints, loss of mobility and significantly impaired quality of life2,3 with an appreciable economic impact on health and social care costs of 1-2% GDP4,5. There is presently no cure for osteoarthritis and current surgical6,7 and pharmacological therapies, whilst being able to give symptomatic relief, do not halt the joint degeneration8-10. Increasing numbers of younger adults (less than 65 years) are presenting to clinicians with advanced cartilage disease or post-traumatic osteoarthritis11. Traumatic injury of the joint tissues (articular cartilage, meniscal cartilage, bone, tendon and ligaments) is one risk factor for the subsequent development of OA12. Over 10,000 cartilage injuries warranting repair occur annually in the UK13 and around 40% of traumatic cartilage lesions will lead to development of OA. Replacement of the osteoarthritic joints with prosthetic joints may ultimately be required to restore mobility. However, problems associated with joint prostheses include a finite working life14 and may not provide the individual with the full range of natural movement as compared to a natural healthy joint. In addition, device loosening is a longer-term problem requiring invasive revision surgery15 and is particularly problematic in younger more active patients (<60 years)16-17. who tend to have an active lifestyle and so put more demands on their prostheses. Therefore, with an increasingly aging population, patients over 50 or 60 years of age may require at least one revisional surgery for knee prostheses.
An aging population and a need to prolong an individual's health and working capacity, coupled with the limitations of artificial joints, indicate a clinical requirement to prolong the functional pain-free life of joints to delay or prevent the need for joint replacement surgery. Currently there are four main approaches to regenerate the joint surface (articular) cartilage before the need for joint replacement. The first approach is the more common approach of using microfracture6,7,18. In this procedure, the surgeon trims the cartilage defect and drills down into the subchondral bone to release bone marrow cells to stimulate regeneration of new cartilage tissue. However, while giving valuable pain relief, a fibrocartilage tissue is formed which is biomechanically inferior to native hyaline cartilage and degenerates after several years. A second, more recent approach, is to place non-functionalised material implants into cartilage defects such as collagen matrices or sponges containing the substitute mineral hydroxyapatite to aid integration of the implant into bone. The third approach covers the developing cell therapy approaches, such as autologous chondrocyte implantation (ACI)19-20 and matrix assisted autologous chondrocyte implantation (MACI)21 which have shown promising results for regeneration of traumatic lesions of articular cartilage on an individual patient basis22-24. Both these cell therapies use autologous chondrocytes isolated from a cartilage biopsy and grown in the laboratory to obtain sufficient numbers for the procedure. Cells are either, placed into the defect area as a cell suspension and then held under a flap of periosteum or a collagen membrane (ACI), or they are embedded in a biomaterial scaffold then implanted (MACI). To date, patients treated with ACI or MACI are less than 50 years of age with cartilage lesions caused by trauma. However, these procedures require the patient to have two separate surgeries and currently, the cost of these technologies does not allow access to a wider patient population. Currently, neither ACI nor MACI are approved by the UK National Centre for Clinical Excellence (NICE)13 so are not funded by the UK National Health Service, unless the patients are participant in a clinical trial investigating the efficacy of these procedures.
There is a clinical need for a cost-effective medical device to actively promote regeneration of the joint surface to delay or prevent joint replacement surgery. Regeneration of articular cartilage in situ requires the appropriate cell homing mechanisms, and retention of ‘repair cells’ [(e.g. mesenchymal stem cells (MSCs)] at the defect site. MSCs are present in the joint fluid (and in raised numbers in OA joint fluids25), and can be readily released from the bone marrow by surgical microfracture of the subchondral bone8,18. The stem cells must then undergo appropriate chondrogenic differentiation and synthesise a hyaline cartilage. It is well known that the extracellular matrix (ECM) of healthy articular cartilage, in common with the ECMs of other body tissues (such as bone), contains sequestered biological/bioactive factors (BFs) such as tissue growth factors, phenotype modulating factors and migratory factors for cell proliferation, differentiation and maintenance of chondrocyte phenotype and tissue integrity6,26,27. These BFs are tightly held within the extracellular matrix or at the cell surface with a large fraction of this potential bioactivity non-covalently bound through charge-charge interactions to sulphated glycosaminoglycan side chains (particularly heparan sulphate) of proteoglycans28. Most sulphated glycosaminoglycans (sGAGs) are present on the cell surface or in the extracellular matrix as extended O-linked side-chains of proteoglycans such as syndecans, perlecan and versican29,30. These sGAGs bind many biologically active molecules non-covalently through ionic interactions. These BFs can be mobilised when required through cell signalling30, and/or proteinase action55 or can act as concentration gradients of chemokines for cell migration31. Regarding the latter, the presence of sGAG-bound chemokines is thought to be crucial for the formation of chemokine gradients essential for migration and homing of stem cells32. The sequestering of bioactive molecules by sGAG moieties maintains their biological activity by sequestering them away from chemical and proteolytic degradation; moreover, sGAGs can enhance the interactions between the cell receptors and biological factor(s)33-35. Some BFs are unstable at body temperature (37° C.) or form oligomerised forms such as TGFβ3 and CXCL12 which are unstable in solution. Interaction of these factors with heparin is essential to induce the oligomerization and stabilisation of the molecular structures36-38. In addition, oligomerisation of some BFs is crucial for full biological function39. Hence, the co-presence of sGAGs and BFs and the resultant ionic binding interaction is important to protect and optimise the biological activity and interaction of many BFs with their target cell receptors and may be crucial for full activity in the body. In addition, sGAGs are often required for the interaction between biological factor and its cell receptor. These activities cannot be fully achieved by direct covalent attachment of bioactive factors to a surface.
Not all BFs found in extracellular matrices, such as native cartilage, bind directly to glycosaminoglycans; for example, the cartilage differentiating and matrix-stimulating factor MIA40 is reported to bind to fibronectin. However, in these cases the bioactive factor may bind to a specific binding protein or an extracellular matrix protein which will bind to glycosaminoglycans. For example, fibronectin binds tightly to sGAG residues and also binds growth factors such as MIA41. Other examples of extracellular matrix proteins which have both growth factor and sGAG-binding sites are vitronectin and laminin42,43.
The GAG sidechains of extracellular matrix proteoglycans are made up of repeating disaccharides with varying degrees of sulphation to form chondroitin sulphate, heparan sulphate, keratan sulphate and dermatan sulphate. It is known that these sulphated oligosaccharides and heparin (a mimic of the GAG, heparan sulphate) can directly bind many BFs, examples of which are bone morphogenic proteins, fibroblast growth factors and the transforming growth factor family of growth factors.
There have been reports of hydrogel and nanoparticle drug delivery systems containing heparin that has been chemically cross-linked to give covalently bound residues, followed by binding of single growth factors such as bone morphogenic protein-2 or fibroblast growth factor-244-47 or vascular endothelial growth factor59. This approach has been used to specifically and covalently bind heparin to a specified collagen scaffold using chemical a cross linker. The cross-linked, heparin-derivitised collagen scaffold was used to bind morphogenic protein-260. However, this report does not show any biological activity of the bound BMP-2 nor any in vitro or in vivo biological activity nor covalent binding of other oligosaccharides or binding of any other BFs to the scaffold. Also, there are reports of various scaffolds which have been soaked in a single growth factor to enable physical absorption to the scaffold surface, or growth factors have been directly bound to a surface or scaffold through a chemically-induced covalent binding or use of streptavidin-labelled growth factors bound to biotin-labelled scaffolds or physical entrapment.
Ionic binding interactions are essential for intercellular, extracellular and intracellular biological reactions needed for life. Such interactions include but are not exclusive to protein-protein interactions and protein-ligand interactions (such as binding of bioactive factors to their target cell receptors and cell signalling), nucleotide-protein interactions and carbohydrate-protein interactions (such as sequestering of bioactive factors by extracellular matrices). Hence, ionic interactions are essential to biochemical, chemical, biomaterial and cell biology methodologies to enable biological mimicry. Ionic interactions are also essential for modification of surfaces to build up alternative layers of anionic and cationic charges, for example layer-by-layer technologies which are well described in the scientific literature47. This approach has been used to bind a protein antibody to the biological factor TGFβ254 to the surface of an artificial lens. It should be noted that this report described the binding of an antibody and not binding of the active biological factor TGFβ2 to the artificial lens surface nor described binding of sulphated oligosaccharides or other sulphated moieties. As predicted by their high negative charge, sulphated glycosaminoglycan moieties (sGAGs) will bind tightly to positively charged surfaces such as those modified with amine groups deposited, for example, by covalent binding, layer-by-layer technologies47 or plasma deposition48-50. Therefore, positively charged surfaces such as amine-modified surfaces can be used to immobilise glycosaminoglycans and oligosaccharides derived therefrom51. Binding of sGAGs through ionic interaction ensures that they are permitted to assume a conformation to enable interaction with BFs and cell receptors. Therefore, heparin-binding can be sequestered to the immobilised glycosaminoglycan oligosaccharides in a form that can be utilised by cells and tissues. Use of just an anionic surface to bind bioactive factors such as chemokines and growth factors, does not permit the co-presence and co-activity of sGAGs and bioactive factors which is an important feature to protect and optimise the biological activity and interaction of many bioactive factors with the target cell receptors and may be crucial for full activity in the body
First reported in 200450, positively charged amine-functionalised glass and plastic surfaces can bind sGAGs through ionic interaction. Heparin bound to the amine-functionalised surfaces of multi-well assay plates has been shown to bind single, known heparin-binding growth factors BMP252, osteoprotogerin, TSG6 and TIMP353 However, these publications did not show biological activity of the bound ligands nor biocompatibility of the functionalised surfaces, nor binding of these biological factors to polymer scaffolds. More recently the same research group (WO 2014/153610)51 described immobilisation of heparin to an amine functionalised cell culture plate and a scaffold of polycaprolactone. The binding of fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor (PDGF), was shown51. for the purpose of using the growth factor immobilisation for in vitro cell culture of epithelial cells, dermal fibroblasts, keratinocytes and retinal pigment epithelial cells and tissue engineering of skin substitutes for wound repair and laboratory skin models. However, WO 2014/15361051 does not report any studies to provide evidence for in vivo activity.
There remains a need for a cell-free medical device for regeneration of skeletal tissues e.g. articular cartilage, meniscus, ligaments and tendons or bone. The invention described hereinafter provides a medical device containing combinations of several bioactive factors to promote in vivo cell homing of mesenchymal stem cells to a cell-free medical device and also promote appropriate cell differentiation and tissue formation. These combinations of bioactive factors can be customised to promote regeneration of articular cartilage, meniscal cartilage, ligaments and tendons or bone.